Cells contain receptors that can elicit a biological response by binding various molecules including proteins, hormones and/or drugs. Nuclear receptors represent a super family of proteins that are hormone/ligand-activated transcription factors that enhance or repress transcription in a cell type-, ligand- and promoter-dependent manner. The nuclear receptor family includes receptors for glucocorticoids (GRs), androgens (ARs), mineralocorticoids (MRs), progestins (PRs), estrogens (ERs), thyroid hormones (TRs), vitamin D (VDRs), retinoids (RARs and RXRs), peroxisomes (XPARs and PPARs) and icosanoids (IRs). The so-called “orphan receptors” for which ligands have not been identified are also part of the nuclear receptor superfamily, as they are structurally homologous to the classic nuclear receptors, such as steroid and thyroid receptors. Although overall sequence conservation between nuclear receptors varies between different families of receptors, sequence conservation between functional regions, or modules, of the receptors is high. For example, nuclear receptors can be organized into functional modules comprising an N-terminal transcriptional activation domain, a central DNA binding domain (DBD), and a C-terminal ligand binding domain (LBD). The LBD of nuclear receptors represents a hormone/ligand-dependent molecular switch. Binding of hormone to a nuclear receptor's LBD changes its ability to modulate transcription of DNA, although they may have transcription-independent actions. Nuclear receptors also bind proteins, such as chaperone complexes, corepressors, or coactivators, that are involved in receptor function. Hormone binding by a nuclear receptor can increase or decrease binding affinity to these proteins, and can influence or mediate the multiple actions of the nuclear receptors on transcription. For example, nuclear receptors can stimulate transcription in response to hormone binding by recruiting coactivator proteins to promoters of responsive genes (Glass et al., Curr. Opin. Cell Biol. (1997) 9:222-32); and Horwitz et al., Mol. Endocrinol. (1996) 10:1167-77).
Coactivators of the p160 family mediate activity of a transcriptional activation domain, called AF2, that is part of the nuclear receptor's LBD. A few receptor mutants deficient in coactivator-dependent activation have been isolated (TR: Collingwood et al. Proc. Natl. Acad. Sci. (1997) 94:248-253; VDR: Jurutka et al., J. Biol. Chem. (1997) 227:14592-14599, Masayama et al., Mol. Endocrinol. (1997) 11:1507-1517; ER and RAR: Henttu et al., Mol. Cell Biol. (1997) 17:1832-1839). While these studies support the physiological relevance of the observed interaction, the structural and functional nature of the site to which coactivators bind has not been defined.
The medical importance of nuclear receptors is significant. They have been implicated in breast cancer, prostate cancer, cardiac arrhythmia, infertility, osteoporosis, hyperthyroidism, hypercholesterolemia, obesity and other conditions. However, limited treatments are available and current agonist/antagonist drugs used to target nuclear receptors are ligands that bind to the receptor's LBD buried deep within the receptor. Although additional targets on nuclear receptors are desired for drug development, the structural and functional basis of such sites, including the coactivator binding site, has not been described.
Accordingly, a need exists for identification and characterization of the coactivator binding sites of nuclear receptors, and molecules that affect their interaction with cellular coactivator proteins. This would provide a major new target for iterative drug design, synthesis, and selection. It also would be advantageous to devise methods and compositions for reducing the time required to discover compounds that target the coactivator binding site of nuclear receptors and administer them to organisms to modulate physiological processes regulated by nuclear receptors.
Relevant Literature
Wagner et al., (Nature (1995) 378:690-697) disclose the crystal structure of rat TR-alpha LBD. Various references disclose mutations in carboxyl-terminal helices of nuclear receptors (Henttu et al., supra; O'Donnell et al., Mol. Endocrinol. (1991) 5:94-99; Whitfield et al., Mol. Endocrinol. (1995) 9:1166-79; Saatcioglu et al., Mol. Cell Biol. (1997) 17:4687-95; Collingwood et al., supra; Kamei et al., Cell (1996) 85:403-14). Hong et al. (Proc. Natl. Acad. Sci. USA (1996) 93(10):498-49452) and Hong et al. (Mol. Cell. Biol. (1997) 17:2735-2744) disclose cloning and expression of GRIP1 coactivator. Torchia et al., (Nature (1997) 387:677-84), Le Douarin et al., (EMBO J (1996) 15:6701-6715) and Heery et al. (Nature (1997) 387:733-736) disclose sequence alignment of various coactivator proteins showing a (SEQ ID NO: 1) LxxLL motif.